Processes of producing glutamic acid compounds and production intermediates therefore and novel intermediate for the processes

ABSTRACT

The present invention relates to processes of producing glutamic acid compounds, for example, monatin, which are useful as, for example, production intermediates for sweetener or pharmaceutical products.

CONTINUING APPLICATION INFORMATION

The present application is a Continuation of International ApplicationNo. PCT/JP02/12473, filed on Nov. 29, 2002, and incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to processes of producing glutamic acidcompounds such as monatin, useful as production intermediates forsweetener or pharmaceutical products, as well as productionintermediates therefore, and a novel important intermediate included inthem. More specifically, the invention relates to a process ofindustrially efficiently producing the glutamic acid compounds, aprocess of producing production intermediates for use therefore and anovel intermediate included in them, and a process of producingoptically active monatin, a process of producing productionintermediates for use therefore, including a novel intermediate.

2. Description of the Background

Glutamic acid compounds such as monatin are compounds that are promisingfor use as sweetener or production intermediates for pharmaceuticalproducts and the like. For example, it has been known that4-hydroxy-4-(3-indolylmethyl)-2-aminoglutaric acid(3-(1-amino-1,3-dicarboxy-3-hydroxybutan-4-yl)indole, sometimes referredto as “monatin” hereinbelow) represented by the following formula (7′)in the (2S,4S) form is contained in the root of a plant Schlerochitonilicifolius and has sweetness at a level several hundreds-fold that ofsucrose (see JP-A-64-25757 (U.S. Pat. No. 4,975,298)).

In the specification, the term “monatin” is not limited to the (2S,4S)form naturally occurring but is used as the generic name of4-hydroxy-4-(3-indolylmethyl)-2-aminoglutaric acid(3-(1-amino-1,3-dicarboxy-3-hydroxybutan-4-yl)indole) including theindividual isomers in the forms of (2S,4S), (2S,4R), (2R,4S), and(2R,4R).

The following reports have been issued about processes of producingmonatin (the following examples (2) to (5)) and protected monatin (thefollowing example (1)).(1) Process described in Tetrahedron Letters, 2001, Vol. 42, No. 39, pp.6793-6796;

(2) Process described in Organic Letters, 2000, Vol. 2, No. 19, pp.2967-2970;

(3) Process described in U.S. Pat. No. 5,994,559;

(4) Process described in Synthetic Communications, 1994, Vol. 24, No.22, pp. 3197-3211;

and(5) Process described in Synthetic Communications, 1993, Vol. 23, No.18, pp. 2511-2526 and U.S. Pat. No. 4,975,298 and U.S. Pat. No.5,128,164;

Because any of the processes requires multiple steps, however, theindustrial practice of the processes actually involves much difficulty.Some of the references shown above or other references (see T. Kitahara,et al., Japanese Agrochemical Association, the 2000-th Conference,Abstracts of Proceedings, 3B128β (p. 221)) describe about theexamination of processes of producing optically active monatin. However,disadvantageously, the processes require multiple steps and involve verytough steps for industrial practice. Thus, it has been desired todevelop an industrial process of efficiently producing glutamic acidcompounds typically including monatin, particularly an industrialprocess of efficiently producing optically active monatin.

SUMMARY OF THE INVENTION

The problems to be solved by the present invention are to provideprocesses of industrially and efficiently producing glutamic acidcompounds such as monatin and production intermediates therefore(including salt forms of them) and to provide important intermediatestherefore. More specifically, the invention provides a process ofindustrially efficiently producing the glutamic acid compounds, aprocess of producing production intermediates for use therefore and anovel important intermediate included in them, and a process ofproducing optically active monatin, a process of producing productionintermediates for use therefore and a novel important intermediateincluded in them.

The inventors have made investigations so as to solve the problemsdescribed above. The inventors have found that glutamic acid compoundssuch as monatin (including salt forms thereof) can be efficientlyproduced by condensing a specific pyruvic acid compound and oxalaceticacid or pyruvic acid together with cross aldol reaction to produceketoglutaric acid compounds as precursors of the intended glutamic acidcompounds and then converting the carbonyl group in the resultingketoglutaric acid compounds to amino group.

In an aldol reaction using carbonyl compounds of different types as inthe present invention, generally, four types of products are produced inmixture through the self aldol reaction of the same types of compoundsand the cross aldol reaction of different types of compounds. Althoughthe self aldol condensation reaction of oxalacetic acid (Journal ofOrganic Chemistry, 1973, Vol. 38, No. 20, pp. 3582-3585) or pyruvic acid(Journal of American Chemical Society, 1964, Vol. 86, pp. 2805-2810;Analytical Chemistry, 1986, Vol. 58, No. 12, pp. 2504-2510) or the crossaldol reaction in a system where one of carbonyl compounds such asglyoxylic acid or oxalacetic acid is never condensed with itself so asingle product can relatively readily be obtained (Tetrahedron Letters,1987, Vol. 28, pp. 1277-1280) has been known so far, no report hasdescribed any example of selectively obtaining a single cross aldolreaction product between oxalacetic acid or pyruvic acid and pyruvicacid compounds.

Additionally, the inventors have found that an optically active monatincan be obtained by reacting a glutaric acid compound of the followingformula (9) with a specific optically active amine to form a diasteromersalt, then crystallizing and separating the resulting diastereomer salt,further dissociating the diastereomer salt or exchanging thediastereomer salt with a different salt to obtain an optically activeglutaric acid compound, then converting the alkoxyimino group (orhydroxyimino group) of the diastereomer salt or the optically activeglutaric acid compound to amino group, crystallizing the resultingmonatin represented by the following formula (13) (racemate at the2-position) in a mixed solvent of water and an organic solvent.

Based on their findings described above, the invention has beenachieved.

Thus, the invention includes inventions relating to the followingproduction processes described below and the novel substance describedbelow in their individual various embodiments.

A process of producing a glutamic acid compound represented by thefollowing formula (7) or a salt thereof, including a step of treating apyruvic acid compound represented by the following formula (1) andoxalacetic acid represented by the following formula (2) by cross aldolreaction and decarboxylation reaction or treating the pyruvic acidcompound (except for pyruvic acid) and a pyruvic acid represented by thefollowing formula (2′) by cross aldol reaction, to obtain a ketoglutaricacid compound represented by the following formula (4) or a saltthereof, and a step of converting the carbonyl group of the ketoglutaricacid compound or a salt thereof to amino group, where the pyruvic acidcompound, oxalacetic acid and pyruvic acid may individually be in saltforms thereof:

in the above formulas, R¹ represents a group selected from alkyl groups,aryl groups, aralkyl groups and heterocyclic ring-containing hydrocarbongroups; and R¹ may have at least one substituent selected from halogenatoms, hydroxyl group, alkyl groups with one to 3 carbon atoms, alkoxygroups with one to 3 carbon atoms and amino group.

A process described above in where the step of converting the carbonylgroup of the ketoglutaric acid compound represented by the formula (4)or a salt thereof to amino group includes a step of reacting an aminecompound represented by the following formula (5) or a salt thereof withthe ketoglutaric acid or a salt thereof, to obtain a glutaric acidcompound represented by the following formula (6) or a salt thereof, anda step of treating the resulting glutaric acid compound or a saltthereof by reducing reaction:

in the above formulas, R¹ represents a group selected from alkyl groups,aryl groups, aralkyl groups and heterocyclic ring-containing hydrocarbongroups; R² represents hydrogen atom or a group selected from alkylgroups, aryl groups and aralkyl groups; and R¹ may have at least onesubstituent selected from halogen atoms, hydroxyl group, alkyl groupswith one to 3 carbon atoms, alkoxy groups with one to 3 carbon atoms andamino group.

A process described above in where the step of converting the carbonylgroup of the ketoglutaric acid represented by the formula (4) or a saltthereof to amino group includes a step of treating the ketoglutaric acidcompound or a salt thereof by reductive animation reaction.

A process described above where the cross aldol reaction is carried outwithin a range of pH 10 to 14.

A process of producing a ketoglutaric acid compound represented by thefollowing formula (4) or a salt thereof, including a step of treating apyruvic acid compound represented by the following formula (1) andoxalacetic acid represented by the following formula (2) by cross aldolreaction and decarboxylation reaction, or treating the pyruvic acidcompound (except for pyruvic acid) and a pyruvic acid represented by thefollowing formula (2′) by cross aldol reaction, where the pyruvic acidcompound, oxalacetic acid and pyruvic acid may individually be in saltforms thereof:

in the above formulas, R¹ represents a group selected from alkyl groups,aryl groups, aralkyl groups and heterocyclic ring-containing hydrocarbongroups; and R¹ may have at least one substituent selected from halogenatoms, hydroxyl group, alkyl groups with one to 3 carbon atoms, alkoxygroups with one to 3 carbon atoms and amino group.

A process described above where the cross aldol reaction is conductedwithin a range of pH 10 to 14.

A process of producing a glutamic acid compound represented by thefollowing formula (7) or a salt thereof, including a step of reacting aketoglutaric acid compound represented by the following formula (4) or asalt thereof with an amine compound represented by the following formula(5) or a salt thereof, to obtain a glutaric acid compound represented bythe following formula (6) or a salt thereof, and a step of treating theresulting glutaric acid compound or a salt thereof by reducing reaction:

in the above formulas, R¹ represents a group selected from alkyl groups,aryl groups, aralkyl groups and heterocyclic ring-containing hydrocarbongroups; R² represents hydrogen atom or a group selected from alkylgroups, aryl groups and aralkyl groups; and R¹ may have at least onesubstituent selected from halogen atoms, hydroxyl group, alkyl groupswith one to 3 carbon atoms, alkoxy groups with one to 3 carbon atoms andamino group.

A process of producing a glutamic acid compound represented by thefollowing formula (7) or a salt thereof, including a step of treating aketoglutaric acid compound represented by the following formula (4) or asalt thereof by reductive amination reaction:

in the above formulas, R¹ represents a group selected from alkyl groups,aryl groups, aralkyl groups and heterocyclic ring-containing hydrocarbongroups; and R¹ may have at least one substituent selected from halogenatoms, hydroxyl group, alkyl groups with one to 3 carbon atoms, alkoxygroups with one to 3 carbon atoms and amino group.

A process of producing monatin represented by the following formula (7′)or a salt thereof, including a step of treating indole-3-pyruvic acidrepresented by the following formula (1′) and oxalacetic acidrepresented by the following formula (2) by cross aldol reaction anddecarboxylation reaction, or treating indole-3-pyruvic acid representedby the following formula (1′) and pyruvic acid represented by thefollowing formula (2′) by cross aldol reaction, to obtain4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid represented by thefollowing formula (4′) or a salt thereof, and a step of converting thecarbonyl group of the ketoglutaric acid or a salt thereof to aminogroup, where indole-3-pyruvic acid, oxalacetic acid and pyruvic acid mayindividually be in salt forms thereof:

A process described above where the step of converting the carbonylgroup of 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid representedby the formula (4′) or a salt thereof to amino group includes a step ofreacting an amine compound represented by the following formula (5) or asalt thereof with the ketoglutaric acid or a salt thereof to obtain aglutaric acid compound represented by the following formula (6′) or asalt thereof, and a step of treating the glutaric acid compound or asalt thereof by reducing reaction:

in the formula, R² represents hydrogen atom, or a substituent selectedfrom alkyl groups, aryl groups and aralkyl groups.

A process described above where the step of converting the carbonylgroup of 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid representedby the formula (4′) or a salt thereof to amino group includes a step oftreating the ketoglutaric acid compound or a salt thereof by reductiveamination reaction.

A process described above where the cross aldol reaction is carried outwithin a range of pH 10 to 14.

A process of producing 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acidrepresented by the following formula (4′) or a salt thereof, including astep of treating indole-3-pyruvic acid represented by the followingformula (1′) and oxalacetic acid represented by the following formula(2) by cross aldol reaction and decarboxylation reaction, or treatingindole-3-pyruvic acid represented by the following formula (1′) andpyruvic acid represented by the following formula (2′) by cross aldolreaction, where indole-3-pyruvic acid, oxalacetic acid and pyruvic acidmay individually be in salt forms thereof:

A process described above where the cross aldol reaction is carried outwithin a range of pH 10 to 14.

A process of producing monatin represented by the following formula (7′)or a salt thereof, including a step of reacting4-hydroxy-4-(3-indolylmethy)-2-ketogluraric acid represented by thefollowing formula (4′) or a salt thereof with an amine compoundrepresented by the following formula (5) or a salt thereof, to obtain aglutaric acid compound represented by the following formula (6′) or asalt thereof, and a step of subsequently treating the glutaric acidcompound or a salt thereof by reducing reaction:

in the formula, R² represents hydrogen atom and a substituent selectedfrom alkyl groups, aryl groups and aralkyl groups.

A process of producing monatin represented by the following formula (7′)or a salt thereof, including a step of treating4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid represented by thefollowing formula (4′) or a salt thereof by reductive aminationreaction:

A process of producing an optically active monatin represented by thefollowing formula (8) or a salt thereof, including the following steps ato c:

in the formula, * denotes an asymmetric center and independentlyrepresents the R- or S-configuration:step a: a step of obtaining an optically active glutaric acid compoundsalt represented by the following formula (11)

in the formula, R², R³, R⁴, R⁵, R⁶ and R⁷ represent the same meanings asdescribed below; in the formula, * denotes an asymmetric center andindependently represents R- or S-configuration

by reacting a glutaric acid compound represented by the followingformula (9)

in the formula, R² represents hydrogen atom, an alkyl group, an arylgroup or an aralkyl group; and the bond marked with wavy line expressesthat both the R-configuration and the S-configuration are included

with an optically active amine represented by the following formula (10)

in the formula, R³, R⁴, R⁵, R⁶ and R⁷ independently represent hydrogenatom or an alkyl group with one to 3 carbon atoms; * denotes anasymmetric center and represents R-configuration or S-configuration,

to form a diastereomer salt, and a step of separating the diastereomersalt by crystallization;step b: a step of generating monatin represented by the followingformula (13) or a salt thereof

in the formula, * denotes an asymmetric center and represents R- orS-configuration; and the bond marked with wavy line means that both theR-configuration and the S-configuration are included,

by dissociating the optically active glutaric acid compound saltrepresented by the formula (11) or exchanging the optically activeglutaric acid compound salt with a different salt, as necessary, toprepare an optically active glutaric acid compound represented by thefollowing formula (12) or a salt thereof (excluding the optically activeglutaric acid compound salt represented by the formula (11))

in the formula, R² represents hydrogen atom, an alkyl group, an arylgroup or an aralkyl group; in the formula, * denotes an asymmetriccenter and represents the R-configuration or the S-configuration,

and a step of converting the alkoxyimino group or hydroxyimino group toamino group;

step c: a step of obtaining an optically active monatin represented bythe formula (8) or a salt thereof by crystallizing monatin representedby the formula (13) or a salt thereof using a mixed solvent of water andan organic solvent.

A process of producing an optically active monatin represented by thefollowing formula (8) or a salt thereof, including the following steps band c:

in the formula, * denotes an asymmetric center and independentlyrepresents the R- or S-configurationstep b: a step of generating monatin represented by the followingformula (13) or a salt thereof

in the formula, * denotes an asymmetric center and represents R- orS-configuration; and the bond marked with wavy line means that both theR-configuration and the S-configuration are included,

by dissociating an optically active glutaric acid compound saltrepresented by the following formula (11)

in the formula, R² represents hydrogen atom, an alkyl group, an arylgroup or an aralkyl group; R³, R⁴, R⁵, R⁶ and R⁷ independently representhydrogen atom or an alkyl group with one to 3 carbon atoms; and *denotes an asymmetric center and independently represents the R- orS-configuration

or exchanging the optically active glutaric acid compound salt with adifferent salt, as necessary, to prepare an optically active glutaricacid compound represented by the following formula (12) or a saltthereof (excluding the optically active glutaric acid compound saltrepresented by the formula (11))

in the formula, R² represents hydrogen atom, an alkyl group, an arylgroup or an aralkyl group; and * denotes an asymmetric center andrepresents the R-configuration or the S-configuration,and by treating the optically active glutaric acid compound by areaction to convert the alkoxyimino group or hydroxyimino group thereofto amino group andstep c: a step of obtaining an optically active monatin represented bythe formula (8) or a salt thereof by crystallizing the monatinrepresented by the formula (13) or a salt thereof using a mixed solventof water and an alcohol.

A process of producing an optically active monatin represented by thefollowing formula (8) or a salt thereof

in the formula, * denotes an asymmetric center and independentlyrepresents the R- or S-configuration,

including a step of crystallizing the salt of monatin represented by thefollowing formula (13) using a mixed solvent of water and alcohol:

in the formula, * denotes an asymmetric center and represents R- orS-configuration; and the bond marked with wavy line means both theR-configuration and the S-configuration are included.

A process of producing an optically active glutaric acid compound saltrepresented by the following formula (11)

in the formula, R² represents hydrogen atom, an alkyl group, an arylgroup or an aralkyl group; R³, R⁴, R⁵, R⁶ and R⁷ independently representhydrogen atom or an alkyl group with one to 3 carbon atoms; and *denotes an asymmetric center and independently represents the R- orS-configuration,

including a step of reacting a glutaric acid compound represented by thefollowing formula (9)

in the formula, R² represents hydrogen atom, an alkyl group, an arylgroup or an aralkyl group; and the bond marked with wavy line means thatboth the R-configuration and S-configuration are included

with an optically active amine represented by the following formula (10)

in the formula, R³, R⁴, R⁵, R⁶ and R⁷ independently represent hydrogenatom or an alkyl group with one to 3 carbon atoms; * denotes anasymmetric center and represents the R- or S-configuration,

to form a diastereomer salt, and a step of separating the diastereomersalt by crystallization.

A process of producing an optically active glutaric acid compoundrepresented by the following formula (12) or a salt thereof (excludingthe optically active glutaric acid compound salt represented by theformula (11))

in the formula, R² represents hydrogen atom, an alkyl group, an arylgroup or an aralkyl group; * denotes an asymmetric center and representsthe R- or S-configuration,

including a step of dissociating an optically active glutaric acidcompound salt represented by the following formula (11)

in the formula, R² represents hydrogen atom, an alkyl group, an arylgroup or an aralkyl group; R³, R⁴, R⁵, R⁶ and R⁷ independently representhydrogen atom or an alkyl group with one to 3 carbon atoms; and *denotes an asymmetric center and independently represents the R- orS-configuration

or exchanging the optically active glutaric acid compound salt with adifferent salt.

A process of producing monatin represented by the following formula (13)or a salt thereof:

in the formula, * denotes an asymmetric center and represents the R- orS-configuration; the bond marked with wavy line expresses that both theR-configuration and the S-configuration are included,

including a step of dissociating an optically active glutaric acidcompound salt represented by the following formula (11)

in the formula, R² represents hydrogen atom, an alkyl group, an arylgroup or an aralkyl group; R³, R⁴, R⁵, R⁶ and R⁷ independently representhydrogen atom or an alkyl group with one to 3 carbon atoms; and *denotes an asymmetric center and independently represents the R- orS-configuration

or exchanging the optically active glutaric acid compound salt with adifferent salt on a needed basis, to prepare an optically activeglutaric acid compound represented by the following formula (12) or asalt thereof (excluding the optically active glutaric acid compound saltrepresented by the formula (11))

in the formula, R² represents hydrogen atom, an alkyl group, an arylgroup or an aralkyl group; * denotes an asymmetric center and representsthe R-configuration or the S-configuration

and a step of treating the resulting optically active glutaric acidcompound by a reaction to convert the alkoxyimino group or hydroxyiminogroup thereof to amino group.

A process of producing monatin represented by the following structuralformula (7′) (including salt forms thereof), the process passing througha process described above:

A compound represented by the following formulas or the formula (4′),(6′), (7″), (11), (12), (14), (15), (16) or (17) (including salt formsthereof), where in the formulas, R² represents hydrogen atom, an alkylgroup, an aryl group or an aralkyl group; R³, R⁴, R⁵, R⁶ and R⁷independently represent hydrogen atom or an alkyl group with one to 3carbon atoms; and * denotes an asymmetric center and represents the R-or S-configuration:

In an embodiment that such a compound is used or prepared in anappropriate salt form in accordance with the invention, the salt form iswith no specific limitation. Such salt form includes for example sodiumsalt, potassium salt, lithium salt, magnesium salt, calcium salt,ammonium salt, and dicyclohexylammonium salt. By salt formation process,desalting process, salt exchange process and the like for routine use sofar, the intended salt can be produced.

DETAILED DESCRIPTION OF THE INVENTION

The more-detailed procedures for carrying out the invention is nowdescribed below.

Production of ketoglutaric acid compound by cross aldol reaction anddecarboxylation reaction between pyruvic acid compound and oxalaceticacid and derivative preparation as glutamic acid compound.

The pyruvic acid compound represented by the following formula (1) andoxalacetic acid represented by the following formula (2) are treated bycross aldol reaction and decarboxylation reaction or the pyruvic acidcompound (excluding pyruvic acid) and pyruvic acid represented by thefollowing formula (2′) are treated by cross aldol reaction, to obtain aketoglutaric acid compound represented by the following formula (4) or asalt thereof, and then, the carbonyl group of the ketoglutaric acidcompound or a salt thereof is converted to amino group, to produce aglutamic acid compound represented by the following formula (7) or asalt thereof. In this case, the pyruvic acid compound, oxalacetic acidand pyruvic acid may individually be in salt forms.

In the formulas, R¹ represents a group selected from alkyl groups, arylgroups, aralkyl groups and heterocyclic ring-containing hydrocarbongroups. These groups may have at least one substituent selected fromhalogen atoms (iodine atom, bromine atom, chlorine atom, fluorine atom,etc.), hydroxyl group, alkyl groups with one to 3 carbon atoms, alkoxygroups with one to 3 carbon atoms and amino group.

As R¹, alkyl groups, aryl groups, aralkyl groups and heterocyclicring-containing hydrocarbon groups with one to 11 carbon atoms (neverincluding the carbon number in substituents if these groups contain thesubstituents) are preferable. For example, R¹ includes alkyl groups suchas isopropyl group, isobutyl group and 1-methylpropyl group, aryl groupssuch as phenyl group and 3-indolyl group, aralkyl groups such as benzylgroup, 2-phenylethyl group and 2-naphthylmethyl group and heterocyclicring-containing hydrocarbon groups such as 3-indolylmethyl group and3-(6-methylindolyl)methyl group.

For the aldol reaction between the pyruvic acid compound represented bythe formula (1) and the pyruvic acid represented by the formula (2′),herein, the case that the pyruvic acid compound represented by theformula (1) is pyruvic acid, namely the case that R¹ is methyl group(the alkyl group with one carbon atom) is never included.

Examples of R¹ with substituents include R¹ having an aromatic ring orheterocyclic ring, provided that the aromatic ring or heterocyclic ringcontains at least one substituent selected from alkyl groups with one to3 carbon atoms, alkoxy groups with one to 3 carbon atoms, and aminogroups. When a benzyl group or 3-indolylmethyl group for example isselected as R¹ in the formula, specifically, the benzene ring or indolering contained in the group may contain at least one substituentselected from halogen atoms (iodine atom, bromine atom, chlorine atom,fluorine atom, etc.), hydroxyl group, alkyl groups with one to 3 carbonatoms, alkoxy groups with one to 3 carbon atoms, and amino group.

In an embodiment where R¹ is 3-indolylmethyl group, in other words, incase that indole-3-pyruvic acid (formula 1′) is used as the pyruvic acidcompound, 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid (formula 4′)or a salt thereof as an intermediate important for monatin productioncan be obtained. Subsequently by converting the carbonyl group of theketoglutaric acid or a salt thereof to amino group, monatin (formula 7′)or a salt thereof can be produced.

Cross Aldol Reaction

The cross aldol reaction is preferably carried out under alkalineconditions. The pyruvic acid compound and oxalacetic acid, or thepyruvic acid compound (excluding pyruvic acid) and pyruvic acid may bepresent in an appropriate solvent for the reaction.

As the reaction solvent, polar solvents such as water, methanol,ethanol, propanol, acetonitrile and dimethylformamide or mixed solventsthereof are preferable. Particularly, water and a mixed solvent (hydrousorganic solvent) of water and polar solvents are preferable.

The pH of the solvent is within a range of preferably 10 to 14, morepreferably 10.5 to 14, still more preferably 11 to 13.

When the pH is too high, the yield is likely to decrease. When the pH istoo low, secondary reactions are likely to take place during the crossaldol reaction.

Bases may satisfactorily be used to achieve such pH under alkalineconditions, and include, for example, inorganic bases such as alkalimetal salts and alkali earth metal salts including alkali earth metalhydroxides and carbonates, e.g., lithium hydroxide, sodium hydroxide,potassium hydroxide, sodium carbonate, potassium carbonate and calciumcarbonate, and organic bases such as triethylamine.

The amount of oxalacetic acid or pyruvic acid to be used to the amountof the pyruvic acid compound has no specific limitation. When oxalaceticacid or pyruvic acid is used in excess, the reaction yield is likely tobe improved. Generally, one to 10 equivalents of oxalacetic acid orpyruvic acid are used to one equivalent of the pyruvic acid compound.Preferably, pyruvic acid can be used within a range of 3 to 6equivalents.

The reaction can be carried at a reaction temperature within a range ofpreferably −10 to 70° C., more preferably 10 to 50° C. When the reactiontemperature is too low, the intended reaction progresses so slowly thatsecondary reactions are likely to occur. When the reaction temperatureis high, the intended ketoglutaric acid compound (or a salt thereof) islikely to be decomposed.

The reaction time has no specific limitation, and is generally one to 72hours, preferably 3 to 24 hours.

Decarboxylation Reaction

The reaction with oxalacetic acid is then progressed to decarboxylationreaction, to subsequently obtain the intended ketoglutaric acid compound(or a salt thereof). The reaction for decarboxylating the condensatefrom the aldol reaction between oxalacetic acid and the pyruvic acidcompound can be achieved by spontaneous decarboxylation reaction.However, the decarboxylation reaction can be effectively progressed byadding an acid or a metal ion or both to the reaction solution. The acidfor use then includes for example hydrochloric acid, sulfuric acid,phosphoric acid, acetic acid, p-toluenesulfonic acid, solid acids suchas ion exchange resins, while the metal ion includes for exampletransition metal ions such as nickel ion, copper ion, and iron ion. Asthe reaction temperature, preferably about −10 to 100° C., morepreferably about 0 to 60° C. can be selected.

The reaction solution after the cross aldol reaction or after the crossaldol reaction and decarboxylation reaction may satisfactorily be usedfor the subsequent step as it is. From the reaction solution theketoglutaric acid compound (or a salt thereof) represented by theformula (4) is isolated and purified, for use in the subsequent step.When the subsequent amination step is continuously carried out,generally, the ketoglutaric acid compound (or a salt thereof) need notbe isolated. After the completion of the reaction, the reaction solutionis concentrated or distilled off, if necessary, for the amination step.By using the same solvent as used in the cross aldol reaction step forthe amination step, the following step can be carried out with nodistillation or solvent substitution of reaction solvent or the like. Incase that the ketoglutaric acid compound represented by the formula (4)is obtained as a salt, the salt is prepared in a free form by a methodknown to a person skilled in the art, for use in the amination step.However, generally, it is not necessary to do so. The salt can be usedin its salt form.

In the cross aldol reaction (and decarboxylation reaction if necessary)in accordance with the invention where R¹ is 3-indolylmethyl group, i.e.where indole-3-pyruvic acid (formula 1′) is used as the pyruvic acidcompound, 4-hydroxy-4-(3-indolylmethyl)-2-ketogluratic acid (formula4′), as an intermediate important for monatin production, or a saltthereof can be produced.

Conversion of the Carbonyl Group to an Amino Group

After the cross aldol reaction is carried out (and subsequently thedecarboxylation reaction is carried out if necessary), the carbonylgroup of the ketoglutaric acid compound represented by the formula (4)or a salt thereof is converted to amino group, to produce a glutamicacid compound represented by the formula (7). The reaction to convertcarbonyl group to amino group is with no specific limitation and iscarried out for example by the following methods.

Conversion Example 1 of a Carbonyl Group to an Amino Group

After the cross aldol reaction is carried out (and subsequently thedecarboxylation reaction is carried out if necessary), an amine compound(which may be in a salt form) represented by the following formula (5)is reacted with the ketoglutaric acid compound represented by theformula (4) or a salt thereof, to produce a glutaric acid compoundrepresented by the formula (6) or a salt thereof, which is then treatedby reducing reaction to produce a glutamic acid compound represented bythe formula (7).

In the formula, R¹ is as described above.

In the case where R¹ is a 3-indolylmethyl group, i.e. in case that4-hydroxy-4-(3-indolylmethyl)-2-ketogluratic acid (formula 4′) is usedas the ketoglutaric acid represented by the formula (4) or a saltthereof, herein, a glutaric acid compound represented by the formula(6′) or a salt thereof is once produced, which is then treated byreduction reaction to produce monatin represented by the formula (7′) ora salt thereof.

In the formula, R² represents a hydrogen atom or a group selected fromalkyl groups, aryl groups and aralkyl groups and the like. R² ispreferably selected from hydrogen atom and alkyl groups and aralkylgroups with 7 or less carbon atoms.

Specifically, R² is preferably a hydrogen atom, methyl group or benzylgroup, particularly preferably hydrogen atom. In other words, specificexamples of the amine compound represented by the formula (5) preferablyinclude hydroxylamine, methoxyamine and benzyloxyamine, particularlypreferably include hydroxylamine.

The salt of the amine compound represented by the formula (5) includessalt forms of the amine compound with organic acids or inorganic acidsand specifically includes for example hydroxylamine hydrochloride salt,hydroxylamine sulfate salt and methoxyamine hydrochloride salt.

In case that hydroxylamine hydrochloride reacts with4-hydroxy-4-(3-indolylmethyl)-2-ketogluratic acid represented by theformula (4′), for example, the corresponding4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminogluratic acid (the compoundof formula (6′) where R² is hydrogen atom) is obtained in good yield.

For the reaction of the ketoglutaric acid compound represented by theformula (4) or a salt thereof with the amine compound represented by theformula (5) or a salt thereof, the reaction temperature can be set atpreferably about −10 to 100° C., more preferably about 0 to 60° C.Further, the reaction time can be set at preferably about one to 100hours, more preferably about 1 to 24 hours.

For the reaction of the amine compound or a salt thereof, the pH of thereaction solution is preferably 2 or more because the reactionprogresses slowly when the pH is too low. The reaction can be progressedmore preferably at about pH 2 to 13, still more preferably at about pH 4to 12.

The ratio of the amine compound or a salt thereof to be used is with notparticularly limited. However, the amine compound (or a salt thereof) isused preferably at about one to 7 moles, more preferably at about one to2 moles per one mole of the ketoglutaric acid compound (or a saltthereof) represented by the formula (4).

As the reaction solvent, polar solvents such as water, methanol,ethanol, propanol, acetonitrile and dimethylformamide, or mixed solventsthereof are preferable. Particularly, water and a mixed solvent of waterwith polar solvents (hydrous organic solvents) are preferable.

As thus obtained, the reaction solution thus obtained, containing theglutaric acid compound (or a salt thereof) represented by the formula(6), may satisfactorily be used as it is for the subsequent step.Otherwise, the glutaric acid compound may be isolated and purified fromthe reaction solution, prior to use in the next step.

When the isolation and purification is carried out, methods known to aperson skilled in the art such as extraction and crystallization canappropriately be used. For example, the glutaric acid compoundrepresented by the formula (6′) may be obtained as follows. That is, byacidifying the reaction solution by adjusting the pH of the reactionsolution with acids such as hydrochloric acid, extracting the glutaricacid compound in organic solvents such as ethyl acetate, concentratingthe resulting organic layer and crystallizing the residue in a mixedsolvent of aqueous ammonia and alcohol, the glutaric acid compoundrepresented by the formula (6′) can be obtained as the diammonium saltthereof in crystals. Using ion exchange resins or adsorption resins orthe like, the compound in the free form can be isolated from thereaction solution. The glutaric acid compound represented by the formula(6′) or a salt thereof as obtained in such manner is generally aracemate, from which the optically active form can be obtained by amethod described below.

Then, the glutaric acid compound represented by the formula (6) (or asalt thereof) is treated by reduction reaction, to produce a glutamicacid compound represented by the following formula (7). Through thereaction, the alkoxyimino group (or hydroxyimino group) at the2-position in the glutaric acid compound represented by the formula (6)can be converted to amino group.

In the formulas, R¹ and R² are as described above.

The reduction of the alkoxyimino group (or hydroxyimino group) to aminogroup can preferably be carried out by hydrogenation reaction using acatalyst for hydrogenation.

As the catalyst for hydrogenation, there can be used palladium-typecatalysts (palladium carbon, etc.), platinum-type catalysts (platinumcarbon, etc.), rhodium-type catalysts (rhodium carbon, etc.),ruthenium-type catalysts (ruthenium carbon, etc.), nickel-type catalysts(Raney-nickel, etc.) and the like.

These catalysts are used within a range of preferably 0.1 to 20 mol %,more preferably 0.5 mol % to 5 mol % of the substrate.

As the reaction solvent, polar solvents such as water, methanol,ethanol, propanol, acetonitrile and dimethylformamide or mixed solventsthereof are preferable. Particularly, water and a mixed solvent (hydrousorganic solvent) of water with polar solvents are preferable.

The reaction of this step is preferably carried out under alkalineconditions, generally within a range of pH 7 to 14, preferably within arange of pH 8 to 12. In the case of using rhodium-type catalysts(rhodium carbon, etc.), in particular, the reaction is carried outgenerally within a range of pH 7.5 to 11, preferably within a range ofpH 8 to 10. In the case of using nickel-type catalysts (Raney nickel,etc.), however, the reaction preferably progresses under neutralconditions, generally within a range of pH 5 to 9, preferably within arange of pH 6.5 to 7.5. If pH is too high, the reaction of this step islikely to give rise to an increase of by-products. If pH is too low, thereaction is likely to progress slowly. In case that the reaction iscarried out under alkaline conditions, the type of an alkali for use inpH adjustment is not specifically limited. For the reduction reactionusing rhodium-type catalysts and palladium-type catalysts, especially,aqueous ammonia is preferably used for the reaction, because of theincrease of the yield and low by-products.

The hydrogenation reaction is preferably carried out in a hydrogenatmosphere. As the hydrogen pressure, a range of preferably 0.5 to 100atmospheres, more preferably 3 to 70 atmospheres, is desirable for thereaction.

The reaction temperature is within a range of preferably −20 to 100° C.,more preferably 0 to 70° C. The reaction time can be 6 to 24 hours.

Conversion Example 2 of the Carbonyl Group to an Amino Group

By converting the carbonyl group at the 2-position in the ketoglutaricacid compound represented by the following formula (4) (or a saltthereof) by reductive amination reaction using amines such as ammonia,benzylamine and 1-phenylethylamine, a glutamic acid compound representedby the following formula (7) can be obtained.

In the formulas, R¹ is as described above.

In case that R¹ is 3-indolylmethyl group, i.e. in case that4-hydroxy-4-(3-indolylmethyl)-2-ketogluratic acid (formula 4′) is usedas the ketoglutaric acid compound represented by the formula (4) or asalt thereof, herein, monatin represented by the formula (7′) or a saltthereof can be produced.

Amine can be used within a range of preferably one to 10 equivalents tothe ketoglutaric acid compound (or a salt thereof). When ammonia is tobe used as the amine, herein, a large excess of ammonia is preferablyused.

As the reducing catalyst, hydride catalysts such as NaBH₄ in addition tothe hydrogenation catalysts described above can be used. In the case ofa hydride catalyst, the catalyst can be used at an amount generallywithin a range of 0.5 to 2 equivalents. In the case of a hydrogenationcatalyst, the catalyst at an amount similar to the amount used forcontact hydrogenation of the glutaric acid compound represented by theformula (6) can be used. The reaction can preferably be progressed at areaction temperature within a range of preferably 0 to 50° C., morepreferably 20 to 35° C. The reaction time is within a range ofpreferably 1 to 72 hours. In case of using a catalyst for hydrogenation,the reaction can be carried out at a hydrogen pressure within 1 to 15atmospheric pressures.

As the reaction solvent, polar solvents such as water, methanol,ethanol, propanol, acetonitrile and dimethylformamide or mixed solventsthereof are preferable. Particularly, water and a mixed solvent (hydrousorganic solvent) of water with polar solvents are preferable.

The glutaric acid compound (or a salt thereof) represented by theformula (7) as obtained by the processes in the two examples can beisolated and purified, using methods known to a person skilled in theart, such as extraction and crystallization. In case that R¹ is3-indolylmethyl group, i.e. in case that4-hydroxy-4-(3-indolylmethyl)-2-ketogluratic acid (formula 4′) is usedas the ketoglutaric acid compound represented by the formula (4) or asalt thereof, herein, monatin represented by the formula (7′) or a saltthereof can be produced. Monatin represented by the formula (7′) or asalt thereof can be isolated and purified by a method for producingoptically active monatin as described below, to obtain the opticallyactive form.

Production of Optically Active Monatin

Monatin has asymmetric carbon atoms at the 2- and 4-positions, so thefollowing four types of optical isomers exist.

In case that 4-hydroxy-4-(3-indolylmethyl)-2-ketogluratic acid (formula4′) is used as the ketoglutaric acid compound represented by the formula(4) or a salt thereof as described above, reaction with an aminecompound represented by the formula (5) or a reagent generating thecompound can produce the glutaric acid compound represented by theformula (6′) or a salt thereof, which is generally a racemate.

The racemate or the glutaric acid compound containing the R-form andS-form at an appropriate ratio (these are included in the glutaric acidcompound represented by the formula (9)) is treated by the followingsteps a to c, to obtain optically active forms of monatin or a saltthereof.step a: a step of obtaining an optically active glutaric acid compoundsalt represented by the following formula (11)

in the formula, R², R³, R⁴, R⁵, R⁶ and R⁷ represent the same meanings asdescribed below; and * denotes an asymmetric center and independentlyrepresents R- or S-configuration,

by reacting a glutaric acid compound represented by the followingformula (9)

in the formula, R² represents hydrogen atom, an alkyl group, an arylgroup or an aralkyl group; and the bond marked with wavy line expressesthat both the R-configuration and the S-configuration are included

with an optically active amine represented by the following formula (10)

in the formula, R³, R⁴, R⁵, R⁶ and R⁷ independently represent hydrogenatom or an alkyl group with one to 3 carbon atoms; and * denotes anasymmetric center and represents R-configuration or S-configuration,

to form a diastereomer salt and then separating the diastereomer salt bycrystallization;step b: a step of generating monatin represented by the followingformula (13) or a salt thereof

in the formula, * denotes an asymmetric center and represents R- orS-configuration; and the bond marked with wave line means that both theR-configuration and the S-configuration are included,

by dissociating the optically active glutaric acid compound saltrepresented by the formula (11) or exchanging the optically activeglutaric acid compound salt with a different salt, as necessary, toprepare an optically active glutaric acid compound represented by thefollowing formula (12) or a salt thereof (excluding the optically activeglutaric acid compound salt represented by the formula (11))

in the formula, R² represents hydrogen atom, an alkyl group, an arylgroup or an aralkyl group; and * denotes an asymmetric center andrepresents the R-configuration or the S-configuration,

and subsequently converting the alkoxyimino group or hydroxyimino groupto amino group;

step c: a step of obtaining an optically active monatin represented bythe formula (8) or a salt thereof by crystallizing monatin representedby the formula (13) or a salt thereof using a mixed solvent of water andan organic solvent.

Step a is described below.

So as to allow the glutaric acid compound represented by the formula (9)to form a diastereomer salt together with the optically active aminerepresented by the formula (10), for example, such compounds or saltsthereof are dissolved in a solvent for the reaction. In case that theglutaric acid compound is in a salt form thereof, the salt is convertedto the free form by neutralizing the salt with an acid as necessary andsubsequently extracting the free form into an organic solvent, and then,the free form reacts with an optically active amine to form the salt.Additionally, an acid may satisfactorily be added to a solventcontaining the glutaric acid compound salt dissolved therein toneutralize the salt, and then, an optically active amine may be addedfor the reaction to form the diastereomer salt. In case that theglutaric acid compound salt represented by the formula (11) is formed bya salt exchange reaction with an optically active amine represented bythe formula (10) in a solvent, the salt form can even be used as is toreact with the optically active amine represented by the formula (10).In this case, the optically active amine represented by the formula (10)is preferably used in a salt form such as hydrochloride salt or sulfatesalt.

Particularly preferable examples of the optically active aminerepresented by the formula (10) include (R)-(+)-1-phenylethylamine and(S)-(−)-1-phenylethylamine, where R³, R⁴, R⁵, R⁶ and R⁷ in the formulaare hydrogen atoms.

The optically active amine is used at an amount of preferably about 0.1to 1-fold mole, more preferably about 0.3 to 0.6-fold that of theglutaric acid compound.

The reaction temperature is set within a range of preferably about −20to 100° C., more preferably about 0 to 60° C. The reaction time is notparticularly limited but is short enough for rapid formation of thesalt.

The reaction solvent includes a single solvent selected from water,methanol, ethanol, acetonitrile, toluene and ethyl acetate, and anappropriate mixed solvent of two or more thereof. Particularly, water ora mixed solvent of water and an organic solvent miscible with water (forexample, polar solvents such as methanol, ethanol and acetonitrile) ispreferably used. Of these, a single solvent of water is more preferable.

After the completion of the reaction, for example, the reaction solutionis concentrated if necessary, and water is added to crystallize thediastereomer salt. The reaction solution may be cooled as necessary.Because water is a poor solvent for the resulting diastereomer salt,water or a mixed solvent of water with an organic solvent miscible withwater is used as the reaction solvent for forming the diastereomer salt,so that the crystal can be deposited and crystallized, concurrently withthe progress of the reaction (salt formation). The crystals obtained bycrystallization are separated from the reaction solution by filtrationand the like, to obtain the diastereomer salt represented by the formula(11). The diastereomer salt obtained as crystals in case of using wateras the poor solvent varies depending on the steric configuration of theoptically active amine used. In the case of using(R)-(+)-1-phenylethylamine as the optically active amine represented bythe formula (10) for the4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid (a compound ofthe formula (9) where R² is hydrogen atom), for example, thediastereomer salt represented by the following formula (14) can beobtained. In the case of using (S)-(−)-1-phenylethylamine, thediastereomer salt represented by the following formula (15) can beobtained.

In the case of using (R)-(+)-1-phenylethylamine, in other words, acrystal of the diastereomer salt can be obtained, where the glutaricacid compound in the S-configuration at the 4-position forms the salttogether with (R)-(+)-1-phenylethylamine. In the case of using(S)-(−)-1-phenylethylamine, a crystal of the diastereomer salt can beobtained, where the glutaric acid compound in the R-configuration at the4-position forms the salt together with (S)-(−)-1-phenylethylamine. Aperson skilled in the art can select an optically active amine suitablefor the intended compound, to form and crystallize such diastereomersalt, so that a diastereomer salt with a desired steric configurationcan be obtained.

Further, after the separation of these diastereomer salts in crystals,the mother liquor contains a glutaric acid compound in a stericconfiguration opposite to the glutaric acid compound separated ascrystals, as its main component. Thus, by adding an optically activeamine with a steric configuration opposite to that of the opticallyactive amine used to form the salt in the mother liquor, to form andcrystallize the diastereomer salt in the same manner as described above,one additional diastereomer salt crystal can be separated from themother liquor. In other words, the step a in accordance with theinvention can be applied to the mother liquor to obtain anotherdiastereomer salt by crystallization as described above.

Step b is described below.

The optically active glutaric acid compound salt represented by theformula (11) as obtained in the step a is dissociated to form anoptically active glutaric acid compound represented by the formula (12),if necessary. Alternatively, the salt can be replaced by a differentsalt (salt exchange), if necessary.

In case of such dissociation and replacement with a different salt (saltexchange), methods known to a person skilled in the art can be used. Thedissociation method is for example a method including a step ofdissolving or suspending the salt in water, alcohol or a mixed solventthereof, a step of neutralizing the resulting solution or suspensionwith acids such as hydrochloric acid or sulfuric acid, and an extractionstep into organic solvents, a method including a step of dissolving thesalt in water, and separating the free form represented by the formula(12) with ion exchange resins or adsorption resins. For isolation of thefree form, the intended isolation can readily be carried out by a knownmethod, for example distillation of a resin eluent solution, or of anextract solution containing the free form under reduced pressure. Incase of the substitution with a different salt, for example, theoptically active glutaric acid compound salt represented by the formula(11) can be dissolved in aqueous alkali metal solutions of sodiumhydroxide and potassium hydroxide and aqueous ammonia solution, fromwhich the free optically active amine is extracted in an organicsolvent, so that the salt can be exchanged. Using a known method such asthe distillation under reduced pressure of the aqueous solution afterextraction or crystallization, additionally, the salt of the opticallyactive glutaric acid compound represented by the formula (12) (excludingthe optically active glutaric acid compound salt represented by theformula (11)) can be isolated.

Monatin represented by the formula (13) can be produced from theoptically active glutaric acid compound represented by the formula (12)thus obtained or from a salt thereof by treating the alkoxyimino group(or hydroxyimino group) thereof with a reaction to convert the group toamino group. Further, monatin represented by the formula (13) can beproduced by treating the alkoxyimino group (or hydroxyimino group) ofthe optically active glutaric acid compound salt represented by theformula (11) with a reaction to convert the group to amino group in thesame manner as described above.

As described above, the conversion of the alkoxyimino group (orhydroxyimino group) to amino group can be carried out by hydrogenationreaction using a catalytic hydrogenation reagent, under reactionconditions as described above.

After the completion of the reaction, the catalyst is removed byfiltration and the like and the filtrate is concentrated, if necessary,from which monatin represented by the formula (13) can be obtained byisolation methods known to a person skilled in the art (for example,crystallization, HPLC, etc.). In an embodiment where the next step c isto be continuously carried out, additionally, monatin represented by theformula (13) need not be isolated, in general. After the completion ofthe reaction, the catalyst is removed from the reaction solution byfiltration and the like. Then, the reaction solution is concentrated ordistilled off, if necessary, to carry out the crystallization accordingto the step c. By using the same solvent as used as the crystallizationsolvent for the hydrogenation reaction, the next step c can be carriedout without distillation of the reaction solvent or solvent replacementor the like. In case that base is used for the hydrogenation reaction,monatin in the reaction solution exists in a salt form thereof. Bytreating the reaction solution after the removal of the catalyst with anion exchange resin and the like, for example, monatin can be prepared asthe free form or can be converted to a different salt (salt exchangeincluding for example the conversion of ammonium salt to sodium salt orpotassium salt or the like). Then, the resulting product can becrystallized. For carrying out the next step c, preferably, theresulting monatin is used in the salt form thereof as it is.

Step c is described below.

Although the monatin represented by the formula (13) or a salt thereofas obtained at the step b can retain the optical activity at the4-position, the resulting monatin can be recovered as a mixture of the Sform and R form at the 2-position. The monatin and a salt thereof can beoptically resolved by crystallization according to the step c describednext, so that optically active monatin or a salt thereof, opticallyactive at both the 2- and 4-positions, can be obtained.

The monatin represented by the formula (13) or a salt thereof is treatedby a crystallization step using a mixed solvent of water and an organicsolvent, to obtain optically active monatin (crystal) represented by theformula (8).

In case that a base is used for the hydrogenation reaction at the step(b), generally, monatin can be obtained generally in a salt formthereof, which is preferably treated at a crystallization step, as it isin the salt form. In this case, water is a good solvent for the monatinsalt. The crystallization method is not particularly limited, andincludes for example methods known to a person skilled in the art, suchas crystallization under cooling and concentration prior tocrystallization. For crystallizing the monatin salt, for example, themonatin crystal in the free form can be obtained by adding an acid to anaqueous solution containing the monatin salt dissolved therein toneutralize the solution and by adding an organic solvent to theresulting solution. Because monatin is likely to be decomposed by anacid, however, the invention can preferably be used particularly in casethat monatin is to be obtained in a salt form.

As the organic solvent, an organic solvent miscible with water can beused. Particularly, alcohols such as methanol, ethanol, propanol andisopropanol are preferable. A mixed solvent of different two types ormore organic solvents may satisfactorily be used as the organic solvent.The ratio of an organic solvent and water in the mixed solvent withwater may satisfactorily be set preferably within a range of an organicsolvent:water=about 1:0.1 to 1:1 in volume ratio, more preferably withina range of an organic solvent:water=about 1:0.3 to 1:0.9 in volumeratio. The crystallization temperature may satisfactorily be set withina range of preferably about −20 to 100° C., more preferably about 0 to60° C.

As shown below in the following schemes, the steric configuration of themonatin crystal obtained at the step c is as follows. In case of usingmonatin represented by the formula (13) in the R form at the 4-positionand in the S form at the 4-position, the monatin crystals (2R,4R) and(2S,4S) are obtained, respectively. Additionally, the mother liquorafter crystal separation individually contain (2S,4R) monatin and(2R,4S) monatin as the main component. By treating the mother solutionswith adsorption resins and the like, (2S,4R) monatin or (2R,4S) monatincan be isolated.

If desired, the optically active monatin obtained in the free form canbe prepared as a salt form. By methods known to a person skilled in theart (salt formation), the monatin can be prepared in salt forms, forexample as a sodium salt or potassium salt. Further, even the opticallyactive monatin obtained in a salt form thereof can be obtained similarlyin the free form if desired or can be converted to a different salt. Byutilizing methods known to a person skilled in the art, for example, thesalt can be converted to the free form by a method for converting saltsto free forms via dissociation and the like. Alternatively, the salt canbe converted to the intended different salt by exchanging the resultingsalt with a different salt (salt exchange).

EXAMPLES

The invention is now described in detail in the following Examples.However, the invention is not limited to the Examples.

In the Examples, further, the optical purity was assayed by HPLC underthe following conditions.

Column for separating optical isomers

SUMICHIRAL OA-7100 manufactured by Sumika Chemical Analysis ServiceEluent

20 mM phosphate buffer, pH 2.8: acetonitrile=7:3

Column temperature

10° C.; and

Flow rate

0.6 ml/min.

Example 1 Synthesis of 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaricacid; NO. 1

After 8.28 g of potassium hydroxide (having a purity of 85% by weight)was dissolved in 27 ml of water, 3.0 g (14.76 mmol) of indole-3-pyruvicacid and 5.85 g (44.29 mmol) of oxalacetic acid were added to theresulting solution, for reaction at ambient temperature for 72 hours(about pH 13 at the start of the reaction). An ion exchange resin(Amberlite IR 120B H AG) was added to the reaction solution to adjustthe solution to pH 3.0, for extraction into 200 ml of ethyl acetate at0° C. 100 ml of saturated aqueous sodium bicarbonate was added to theresulting ethyl acetate layer, and ethyl acetate in the ethyl acetatelayer was distilled off, and pH of the solution was re-adjusted to 7.9with an ion exchange resin (IRA400 OH AG manufactured by OrganoCorporation). The resulting solution was freeze-dried as it was4-Hydroxy-4-(3-indolylmethyl)-2-ketoglutarate sodium salt was obtainedas a crude product. Further, 40 ml of water and 200 ml of ethanol wereadded to the resulting residue, in which the solid was filtered off. Theresulting mother solution was concentrated to dryness, to obtain4-hydroxy-4-(3-indolylmethyl)-2-ketoglutarate sodium salt of 1.5 g as acrude product.

Example 2 Synthesis of 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaricacid; NO. 2

After 18.91 g (286.5 mmol) of potassium hydroxide (at a content of 85%by weight) was dissolved in 64.45 ml of water, 7.50 g (35.8 mmol at acontent of 97.0% by weight) of indole-3-pyruvic acid and 14.18 g (107.4mmol) of oxalacetic acid were added to and dissolved in the resultingsolution (about pH 13 at the start of the reaction). The mixed solutionwas stirred at 35° C. for 24 hours. Further, 40.0 ml of 3N hydrochloricacid was added for neutralization (pH=7.0), to obtain 153.5 g of aneutralized reaction solution. The neutralized reaction solutioncontained 5.55 g of 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid ata yield of 53.5% (vs. indole-3-pyruvic acid). Water was added to theneutralized reaction solution to make up 168 ml, and the solution passedthrough a resin column (diameter of 4.8 cm) packed with a syntheticadsorbent (DIAION-SP207 manufactured by Mitsubishi Chemical Corporation)of 840 ml. Further, pure water was passed through the column at a flowrate of 23.5 ml per minute. 1.73 to 2.55 (L/L-R) were collected, toobtain an aqueous solution of4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid of 3.04 g at a yieldof 54.7% (to the charged amount in the resin).

(NMR Measurement)

¹H-NMR (400 MHz, D₂O): δ 3.03 (d, 1H, J=14.6 Hz), 3.11 (d, 1H, J=14.6Hz), 3.21 (d, 1H, J=18.1 Hz), 3.40 (d, 1H, J=18.1 Hz), 7.06-7.15 (m,3H), 7.39 (d, 1H, J=7.8 Hz), 7.66 (d, 1H, J=7.8 Hz).

¹³C-NMR (400 MHz, D₂O): δ 35.43, 47.91, 77.28, 109.49, 112.05, 119.44,119.67, 121.91, 125.42, 128.41, 136.21, 169.78, 181.43, 203.58.

(Molecular Weight Measurement)

Theoretical ESI-MS value C₁₄H₁₃NO₆=291.07

Analytical value=290.02 (M-H)⁻

Example 3 Synthesis of 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaricacid; NO. 3

After 3.70 g (56.0 mmol) of potassium hydroxide (at a content of 85% byweight) was dissolved in 72.1 ml of water, 0.81 g (4.0 mmol) ofindole-3-pyruvic acid and 3.17 g (24.0 mmol) of oxalacetic acid wereadded to and dissolved in the resulting solution (about pH 13 at thestart of the reaction). The mixed solution was stirred at 35° C. for 24hours. A part of the reaction solution was treated with hydroxylamine toprepare 4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid, whichwas analyzed by HPLC. Consequently, it was found that4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid was generated at ayield of 76.6% (vs. indole-3-pyruvic acid).

Example 4 Synthesis of 4-benzyl-4-hydroxy-2-ketoglutaric acid

After 16.23 g of potassium hydroxide (having a purity of 85% by weight)was dissolved in 48 ml of water, 5.0 g (30.5 mmol) of phenylpyruvic acidand 12.1 g (91.4 mmol) of oxalacetic acid were added to the resultingsolution, for reaction at ambient temperature for 72 hours (about pH 13at the start of the reaction). Using conc. hydrochloric acid, thereaction solution was adjusted to pH 2.2, and extracted in ethylacetate. The organic layer was rinsed in aqueous saturated sodiumchloride, dried over anhydrous magnesium sulfate and concentrated toobtain the residue. The residue was recrystallized in ethyl acetate andtoluene, to obtain 2.8 g (11.3 mmol) of4-benzyl-4-hydroxy-2-ketoglutaric acid in crystal form.

(NMR Measurement)

¹H NMR (D₂O) δ 2.48 (d, J=14.4 Hz, 0.18H), 2.60 (d, J=14.4 Hz, 0.18H),2.85-3.30 (m, 3.64H), 7.17-7.36 (m, 5H).

(Molecular Weight Measurement)

Theoretical ESI-MS value C₁₂H₁₂O₆=252.23

Analytical value=251.22 (M-H)⁻

Example 5 Synthesis of4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid; NO. 1

After 13.8 g of potassium hydroxide (having a purity of 85% by weight)was dissolved in 50 ml of water, 5.0 g (24.6 mmol) of indole-3-pyruvicacid and 9.8 g (73.8 mmol) of oxalacetic acid were added to theresulting solution, for reaction at ambient temperature for 72 hours(about pH 13 at the start of the reaction). To the reaction solution wasadded 6.8 g (98.4 mmol) of hydroxylamine hydrochloride salt. Then, thereaction solution was adjusted to pH 7.5 with aqueous 4N sodiumhydroxide solution. After the reaction solution had been stirred for 24hours at ambient temperature, the reaction solution was adjusted to pH2.6 with 6N hydrochloric acid. After extraction using ethyl acetate, theorganic layer was rinsed in aqueous saturated sodium chloride, driedover anhydrous magnesium sulfate and concentrated to dryness. Theresulting residue was dissolved in 10 ml of aqueous 14% ammonia,followed by gradual dropwise addition of 70 ml of ethanol, and stirredat ambient temperature for 3 hours. The resulting slurry was filtered.The resulting crystals was dried to obtain 2.7 g (7.9 mmol) of4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid in the form ofan ammonium salt.

(NMR Measurement)

¹H NMR (DMSO-d₆) δ: 2.66 (s, 2H), 2.89 (d, J=14.4 Hz, 1H), 3.04 (d,J=14.4 Hz, 1H), 6.89-6.94 (m, 1H), 6.97-7.03 (m, 1H), 7.11 (d, J=2.8 Hz,1H), 7.27 (d, J=7.8 Hz, 1H), 7.53 (d, J=7.8 Hz, 1H), 10.71 (br s, 1H).

(Molecular Weight Measurement)

Theoretical ESI-MS value C₁₄H₁₄N₂O₆=306.28

Analytical value=305.17 (M-H)⁻

Example 6 Synthesis of4-hydroxy-4-(3-indolylmethyl)-2-methoxyiminoglutaric acid

After 9.12 g (138.1 mmol) of potassium hydroxide (at a content of 85% byweight) was dissolved in 23 ml of water, 2.55 g (12.2 mmol at a contentof 97.0% by weight) of indole-3-pyruvic acid and 7.46 g (56.5 mmol) ofoxaloacetic acid were added to and dissolved in the resulting solution(about pH 13 at the start of the reaction). The solution was stirred at35° C. for 24 hours. To the reaction solution was gradually added 5.76 g(69 mmol) of methoxyamine hydrochloride salt while the reaction solutionwas adjusted to around pH 10 with aqueous 25% sodium hydroxide solution.After the reaction had continued at ambient temperature for 14 hours,the reaction solution was adjusted to pH 2.23, using 6N hydrochloricacid, and was then extracted in ethyl acetate. The organic layer wasrinsed with aqueous saturated sodium chloride and dried over anhydrousmagnesium sulfate. After the magnesium sulfate had been filtered off,the resulting solution was concentrated to obtain 4.66 g of residue. Theresulting residue was approximately purified by silica gel columnchromatography, and further coarsely purified by preparative thin layerchromatography (PTLC; ethyl acetate/hexane/acetic acid=5/5/1), to obtain0.93 g of the title compound4-hydroxy-4-(3-indolylmethyl)-2-methoxyiminoglutaric acid (2.92 mmol;yield, 24% (vs. indole-3-pyruvic acid)).

(NMR Measurement)

¹H-NMR (400 MHz, DMSO-d₆): δ 2.89 (d, J=14.9 Hz, 1H), 3.04 (s, 2H), 3.15(d, J=14.9 Hz, 1H), 3.90 (s, 3H), 6.91-6.96 (m, 1H), 6.98-7.04 (m, 1H),7.09-7.12 (m, 1H), 7.29 (d, J=7.4 Hz, 1H), 7.50 (d, J=7.4 Hz, 1H), 10.80(br s, 1H)

Example 7 Synthesis of 4-benzyl-4-hydroxy-2-hydroxyiminoglutaric acid

After 16.23 g (having a purity of 85% by weight) of potassium hydroxidewas dissolved in 45 ml of water, 5.0 g (30.5 mmol) of phenylpyruvic acidand 12.1 g (91.4 mmol) of oxalacetic acid were added to the resultingsolution, for reaction at ambient temperature for 24 hours (about pH 13at the start of the reaction). To the reaction solution was added 8.5 g(121.8 mmol) of hydroxylamine hydrochloride salt, for reaction atambient temperature for 72 hours. The reaction solution was adjusted topH 2.6, using 6N hydrochloric acid, and was then extracted in ethylacetate. The organic layer was rinsed with aqueous saturated sodiumchloride, dried over anhydrous magnesium sulfate and concentrated todryness. The resulting residue was recrystallized in 20 ml of ethylacetate and 80 ml of toluene, to obtain 4.0 g (15.1 mmol) of4-benzyl-4-hydroxy-2-hydroxyiminoglutaric acid.

(NMR Measurement)

¹H NMR (DMSO-d₆) δ: 2.80 (d, J=13.9 Hz, 1H), 2.99 (d, J=12.7 Hz, 1H),3.01 (d, J=13.9 Hz, 1H), 3.03 (d, J=12.7 Hz, 1H), 7.13-7.25 (m, 5H).

(Molecular Weight Measurement)

Theoretical ESI-MS value C₁₂H₁₃NO₆=267.24

Analytical value=266.12 (M-H)⁻

Example 8 Synthesis of 4-hydroxy-4-(3-indolylmethyl)-2-aminoglutaricacid (monatin); NO. 1

0.13 g (0.38 mmol) of4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid ammonium saltwas dissolved in 5 ml of aqueous 28% ammonia, to which 0.09 g of 5%rhodium carbon was added, for reaction at ambient temperature and ahydrogen pressure of 7.5 atmospheres. 14 hours later, the catalyst wasfiltered off, and the resulting solution was concentrated to dryness, toobtain a mixture of 0.075 g (0.23 mmol) of the ammonium salt of(2S,4S)/(2R,4R)-4-hydroxy-4-(3-indolylmethyl)-2-aminoglutaric acid(monatin) and 0.036 g (0.11 mmol) of the ammonium salt of(2S,4R)/(2R,4S)-4-hydroxy-4-(3-indolylmethyl)-2-aminoglutaric acid(monatin).

(NMR Measurement)

¹H NMR (D₂O) δ: 2.05 (dd, J=12.2, 15.1 Hz, 0.67H), 2.21 (dd, J=9.9, 15.6Hz, 0.33H), 2.48 (dd, J=3.2, 15.6 Hz, 0.33H), 2.68 (dd, J=2.2, 15.1 Hz,0.67H), 3.08 (d, J=14.4 Hz, 0.67H), 3.17-3.25 (m, 0.66H), 3.28 (d,J=14.4 Hz, 0.67H), 3.63 (dd, J=2.2, 12.2 Hz, 0.67H), 3.98 (dd, J=3.2,9.9 Hz, 0.33H), 7.12-7.18 (m, 1H), 7.19-7.26 (m, 2H), 7.45-7.51 (m, 1H),7.70-7.76 (m, 1H).

(Molecular Weight Measurement)

Theoretical ESI-MS value C₁₄H₁₆N₂O₅=292.29

Analytical value=291.28 (M-H)⁻

Example 9 Synthesis of 4-hydroxy-4-(3-indolylmethyl)-2-aminoglutaricacid (monatin); NO. 2

0.264 g (0.824 mmol) of4-hydroxy-4-(3-indolylmethyl)-2-methoxyiminoglutaric acid was dissolvedin 10 ml of aqueous 28% ammonia. 0.18 g of 5% rhodium carbon (dryproduct) was added and the mixture stirred at a hydrogen pressure of 7.5atmospheres for 18 hours. The catalyst was filtered off and the solventwas distilled off under reduced pressure, to obtain the residue. Theresulting residue was analyzed by NMR, and a mixture of 0.115 g (0.395mmol; yield, 48%) of(2S,4S)/(2R,4R)-4-hydroxy-4-(3-indolylmethyl)-2-aminoglutaric acid(monatin) and 0.065 g (0.223 mmol; yield, 27%) of(2S,4R)/(2R,4S)-4-hydroxy-4-(3-indolylmethyl)-2-aminoglutaric acid wasfound to have been generated.

Example 10 Synthesis of 4-hydroxy-4-(3-indolylmethyl)-2-aminoglutaricacid (monatin); NO. 3

1.0 g (2.94 mmol) of4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutarate ammonium salt wasdissolved in 10 ml of water, to which 1 ml of a Raney-nickel catalyst(manufactured by Kawaken Fine Chemicals Co., Ltd.; developed nickelcatalyst NDHT-90) was added with a syringe, and the mixture stirred at ahydrogen pressure of 20 atmospheres for 10 hours. The catalyst wasfiltered off, and the resulting solution was concentrated to obtain theresidue. The residue was analyzed by NMR. It was shown that 0.29 g (0.89mmol; yield, 30%) of(2S,4S)/(2R,4R)-4-hydroxy-4-(3-indolylmethyl)-2-aminoglutaric acid(monatin) and 0.29 g (0.89 mmol; yield, 30%) of(2S,4R)/(2R,4S)-4-hydroxy-4-(3-indolylmethyl)-2-aminoglutaric acid weregenerated.

Example 11 Synthesis of 2-amino-4-benzyl-4-hydroxyglutaric acid; NO. 1

0.25 g (0.94 mmol) of 4-benzyl-4-hydroxy-2-hydroxyiminoglutaric acid wasdissolved in 10 ml of aqueous 50% methanol solution, to which 0.5 ml ofaqueous 28% ammonia was added. 1.0 g of 5% palladium-carbon (50% hydrousproduct) was added, for reaction at ambient temperature and a hydrogenpressure of 7.7 atmospheres. 72 hours later, the catalyst was filteredoff, and the reaction solution was concentrated to dryness, to obtain0.10 g (0.35 mmol) of the ammonium salt of(2S,4S)/(2R,4R)-2-amino-4-benzyl-4-hydroxyglutaric acid and 0.10 g (0.35mmol) of the ammonium salt of(2R,4S)/(2S,4R)-2-amino-4-benzyl-4-hydroxyglutaric acid as a mixture.

(NMR Measurement)

¹H NMR (D₂O) δ: 1.94 (dd, J=11.9, 15.3 Hz, 0.5H), 2.10 (dd, J=10.2, 15.3Hz, 0.5H), 2.36 (dd, J=3.1, 15.3 Hz, 0.5H), 2.56 (dd, J=2.4, 15.3 Hz,0.5H), 2.81 (d, J=13.6 Hz, 0.5H), 2.94 (d, J=13.5 Hz, 0.5H), 3.01 (d,J=13.5 Hz, 0.5H), 3.06 (d, J=13.6 Hz, 0.5H), 3.55 (dd, J=2.4, 11.9 Hz,0.5H), 3.88 (dd, J=3.1, 10.2 Hz, 0.5H), 7.17-7.31 (m, 5H)

(Molecular Weight Measurement)

Theoretical ESI-MS value C₁₂H₁₅NO₅=253.26

Analytical value=252.23 (M-H)⁻

Example 12 Synthesis of 2-amino-4-benzyl-4-hydroxyglutaric acid; NO. 2

0.13 g (0.52 mmol) of 4-benzyl-4-hydroxy-2-ketoglutaric acid and 0.11 ml(1.0 mmol) of benzylamine were dissolved in 5 ml of methanol, to which0.1 g of 5% palladium carbon (50% hydrous product) was added, forreaction under hydrogen atmosphere at ambient temperature andatmospheric pressure. Two days later, the catalyst was filtered off, andthe reaction solution was concentrated to dryness, to obtain 0.03 g(0.12 mmol) of (2S,4S)/(2R,4R)-2-amino-4-benzyl-4-hydroxyglutaric acidand 0.06 g (0.24 mmol) of(2R,4S)/(2S,4R)-2-amino-4-benzyl-4-hydroxyglutaric acid as a mixture.

Example 13 Synthesis of4-hydroxy-4-(4-hydroxyphenylmethyl)-2-hydroxyiminoglutaric acid

To 10 ml of water in which were dissolved 3.18 g of potassium hydroxide,were added 1.0 g (5.55 mmol) of 4-hydroxyphenylpyruvic acid and 2.2 g(16.7 mmol) of oxalacetic acid, for reaction at ambient temperature for72 hours (about pH 13 at the start of the reaction). Hydroxylaminehydrochloride salt of 1.54 g (22.2 mmol) was added to the reactionsolution, for reaction at ambient temperature for 10 hours. The reactionsolution was then adjusted to pH 2.6, using 6N hydrochloric acid, forextraction using ethyl acetate. The organic layer was rinsed withaqueous saturated sodium chloride, dried over anhydrous magnesiumsulfate and concentrated to dryness, to obtain 0.7 g (2.47 mmol) of4-hydroxy-4-(4-hydroxyphenylmethyl)-2-hydroxyiminoglutaric acid as acrude product. Further, the crude product was recrystallized in methanoland toluene, to obtain 0.22 g (0.78 mmol) of4-hydroxy-4-(4-hydroxyphenylmethyl)-2-hydroxyiminoglutaric acid incrystal.

(NMR Measurement)

¹H NMR (DMSO-d₆) δ 2.67 (d, J=13.7 Hz, 1H), 2.89 (d, J=13.7 Hz, 1H),2.95 (d, J=12.5 Hz, 1H), 2.99 (d, J=12.5 Hz, 1H), 6.59 (d, J=8.0 Hz,2H), 6.97 (d, J=8.0 Hz, 2H), 9.11 (br s, 1H).

(Molecular Weight Measurement)

Theoretical ESI-MS value C₁₂H₁₃NO₆=283.24

Analytical value=281.93 (M-H)⁻

Example 14 Synthesis of4-hydroxy-4-(4-hydroxyphenylmethyl)-2-hydroxyiminoglutarate acid

0.06 g (0.21 mmol) of4-hydroxy-4-(4-hydroxyphenylmethyl)-2-hydroxyiminoglutarate acid wasdissolved in 2.5 ml of aqueous 28% ammonia, to which 0.04 g of 5%rhodium carbon was added for reaction at ambient temperature and ahydrogen pressure of 7.5 atmospheres. 14 hours later, the catalyst wasfiltered off, and the resulting solution was concentrated to dryness, toobtain a mixture of 0.044 g (0.145 mmol) of(2S,4S)/(2R,4R)-4-hydroxy-4-(4-hydroxyphenylmethyl)-2-aminoglutaric acidand 0.021 g (0.069 mmol) of(2S,4R)/(2R,4S)-4-hydroxy-4-(4-hydroxyphenylmethyl)-2-aminoglutaricacid.

¹H NMR (D₂O) δ: 1.89 (dd, J=11.9, 15.7 Hz, 0.68H), 2.06 (dd, J=10.2,15.0 Hz, 0.32H), 2.30 (dd, J=3.3, 15.0 Hz, 0.32H), 2.51 (dd, J=2.4, 15.7Hz, 0.68H), 2.70 (d, J=13.4 Hz, 0.68H), 2.83 (d, J=13.4 Hz, 0.32H), 2.90(d, J=13.4 Hz, 0.32H), 2.96 (d, J=13.4 Hz, 0.68H), 3.52 (dd, J=2.4, 11.9Hz, 0.68H), 3.84 (dd, J=3.3, 10.2 Hz, 0.32H), 6.71-6.77 (m, 2H),7.02-7.08 (m, 2H).

(Molecular Weight Measurement)

Theoretical ESI-MS value Cl₂H₁₅NO₆=269.26

Analytical value=268.11 (M-H)⁻

Example 15 Synthesis of 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaricacid; NO. 4

12.30 g (58.7 mmol; at a purity of 97.0% by weight) of indole pyruvicacid was added to and dissolved in 209 ml of water containing 2.45 g ofsodium hydroxide dissolved therein. Over a period of two hours 47.61 gof an aqueous 25% by weight sodium hydroxide solution and a mixture of25.85 g (293.5 mmol) of pyruvic acid and 25.85 g of water were added tothe resulting solution under a nitrogen atmosphere at 35° C., while thereaction system was kept at pH 11.0. Subsequently, the reaction systemwas agitated for 14 hours. In this way, a reaction solution wasobtained, which contained 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaricacid at a yield of 44.1% (vs. indolepyruvic acid). 3.60 g of 1Nhydrochloric acid was added to the solution for neutralization(pH=6.91), to obtain 275 ml of a neutralized reaction solution.

168 ml of the thus obtained neutralized reaction solution was passedthrough a resin column (having a diameter of 4.8 cm) packed with 840 mlof a synthetic adsorbent (DIAION-SP207 manufactured by MitsubishiChemical Corporation). Then, pure water was passed through the column ata flow rate of 23.5 ml per minute, to collect 1.7 to 2.9 (L/L-R) toobtain 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid at a highpurity at a yield of 66.3%.

(NMR Spectrum)

¹H-NMR (400 MHz, D₂O): δ 3.03 (d, 1H, J=14.6 Hz), 3.11 (d, 11, J=14.6Hz), 3.21 (d, 1H, J=18.1 Hz), 3.40 (d, 1H, J=18.1 Hz), 7.06-7.15 (m,3H), 7.39 (d, 1H, J=7.8 Hz), 7.66 (d, 1H, J=7.8 Hz).

¹³C-NMR (400 MHz, D₂O): δ35.43, 47.91, 77.28, 109.49, 112.05, 119.44,119.67, 121.91, 125.42, 128.41, 136.21, 169.78, 181.43, 203.58.

(Mass Analysis)

Theoretical ESI-MS value C₁₄H₁₃NO₆=291.07

Analytical value=290.02 (M-H)⁻

Example 16 Synthesis of4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid; NO. 2

After 1.0 g (4.92 mmol) of indole-3-pyruvic acid was added to anddissolved in 10 ml of aqueous saturated sodium carbonate solution, theresulting solution was adjusted to pH 12.55 using aqueous 25% sodiumhydroxide solution. After 1.3 g (14.8 mmol) of pyruvic acid was added,the resulting solution was adjusted to pH 12.6 using aqueous 25% sodiumhydroxide solution, for reaction at ambient temperature for 2 hours, toobtain a reaction solution containing4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid. 1.37 g (19.7 mmol) ofhydroxylamine hydrochloride salt was added to the reaction solution,while the reaction solution was adjusted to a pH value around neutralityusing aqueous 25% sodium hydroxide solution, and stirred at ambienttemperature for 4 hours. Using conc. hydrochloric acid, the reactionsolution was adjusted to an acidic pH value, to extract the organicmatter in ethyl acetate. The organic layer was rinsed with aqueoussaturated sodium chloride, dried over anhydrous magnesium sulfate andsubsequently concentrated, to obtain the residue. The residue wasrecrystallized in aqueous 28% ammonia and ethanol, to obtain 0.52 g of4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid (1.5 mmol; ayield, 31% vs. indole-3-pyruvic acid) in crystal form.

(NMR Spectrum)

¹HNMR (DMSO-d₆) δ: 2.66 (s, 2H), 2.89 (d, J=14.4 Hz, 1H), 3.04 (d,J=14.4 Hz, 1H), 6.89-6.94 (m, 1H), 6.97-7.03 (m, 1H), 7.11 (d, J=2.8 Hz,1H), 7.27 (d, J=7.8 Hz, 1H), 7.53 (d, J=7.8 Hz, 1H), 10.71 (br s, 1H).

(Mass Analysis)

Theoretical ESI-MS value C₁₄H₁₄N₂O₆=306.28

Analytical value=305.17 (M-H)⁻

Example 17 Synthesis of4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid; NO. 3

After 10.0 g (49.2 mmol) of indole-3-pyruvic acid was added to anddissolved in 98 ml of aqueous saturated sodium carbonate solution, theresulting solution was adjusted to pH 12.4 using aqueous 25% sodiumhydroxide solution. After 16.3 g (147.6 mmol) of sodium pyruvate wasadded for reaction at ambient temperature for 2 hours, a reactionsolution containing 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acidwas obtained. 13.7 g (197 mmol) of hydroxylamine hydrochloride salt wasadded to the reaction solution while the reaction solution was adjustedto a pH value around neutrality using aqueous 25% sodium hydroxidesolution, and agitated at ambient temperature for 4 hours. Using conc.hydrochloric acid, the reaction solution was adjusted to an acidic pHvalue, to extract the organic matter in ethyl acetate. The organic layerwas rinsed with aqueous saturated sodium chloride, dried over anhydrousmagnesium sulfate and subsequently concentrated, to obtain the residue.The residue was recrystallized in aqueous 28% ammonia and ethanol, toobtain 5.51 g of 4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaricacid (16.2 mmol; a yield of 32% vs. indole-3-pyruvic acid) in crystalform.

Example 18

After 1.0 g (4.92 mmol) of indole-3-pyruvic acid was added to anddissolved in 10 ml of aqueous saturated sodium carbonate solution in thesame manner as in Example 16, the resulting solution was adjusted to pH12.7 using aqueous 25% sodium hydroxide solution. After 1.3 g (14.8mmol) of pyruvic acid was added, the resulting solution was adjusted topH 10.0 using aqueous 25% sodium hydroxide solution, for reaction atambient temperature for 6 hours. 1.37 g (19.7 mmol) of hydroxylaminehydrochloride salt was added to the reaction solution while the reactionsolution was adjusted to a pH value around neutrality using aqueous 25%sodium hydroxide solution, and stirred at ambient temperature for 13hours. Using conc. hydrochloric acid, the reaction solution was adjustedto an acidic pH value, to extract the organic matter in ethyl acetate.The organic layer was rinsed with aqueous saturated sodium chloride,dried over anhydrous magnesium sulfate and subsequently concentrated, toobtain the residue. The residue was analyzed by HPLC, by which it wasshown that 4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid wasgenerated at a yield of about 14%.

Example 19 Synthesis of4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid; NO. 4

73.8 g (352 mmol) of indole-3-pyruvic acid was added to and dissolved in917 g of aqueous 1.6 wt % sodium hydroxide solution. The resultingsolution was adjusted to 35° C., to which 310.2 g (1761 mmol) of aqueous50% pyruvic acid solution was added dropwise over 2 hours, while thereaction solution was retained at pH 11.1 using aqueous 30% sodiumhydroxide solution. After reaction for another 4.5 hours, a reactionsolution containing 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acidwas obtained. 367.2 g (2114 mmol) of aqueous 40% hydroxylaminehydrochloride salt solution was added to the reaction solution while thereaction solution was kept at pH 7 using aqueous 30% sodium hydroxidesolution, and stirred at 5° C. for 17.5 hours. Using conc. hydrochloricacid, the reaction solution was adjusted to pH 2, to extract the organicmatter in ethyl acetate. The organic layer was rinsed with aqueoussaturated sodium chloride and concentrated, to obtain the residue. Theresidue was recrystallized in 60 ml of aqueous 28% ammonia and 1350 mlof 2-propanol, to obtain 43.4 g of the diammonium salt of4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid (142 mmol; ayield of 40% vs. indole-3-pyruvic acid) in crystal form.

Example 20 Production of (R)-(+)-1-phenylethylamine salt of(4S)-4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid

After 44.7 g (0.131 mol) of the ammonium salt of4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid was dissolvedin 500 ml of water at 25° C., the resulting aqueous solution wasadjusted to pH 2 using 25.5 g of 36% hydrochloric acid. The acidicsolution was extracted in 1300 ml of ethyl acetate, and the resultingethyl acetate solution was rinsed with 200 ml of aqueous saturatedsodium chloride solution. 500 ml of an aqueous sodium carbonate solution(13.9 g (0.131 mole) of sodium carbonate) was added to the resultingethyl acetate solution for agitation, to separate the aqueous alkalisolution from ethyl acetate. 23.1 g of 36% hydrochloric acid was addedto the resulting aqueous alkali solution, to adjust the solution to pH2. 6.99 g (57.6 mmol) of (R)-(+)-1-phenylethylamine was added dropwiseto the resulting aqueous acidic solution and the solution stirred at 25°C. for one hour. The resulting crystal was filtered and dried underreduced pressure, to obtain 21.8 g (47.8 mmol) of the(R)-(+)-1-phenylethylamine salt of(4S)-4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid. (Yield,72.7%; optical purity, 87.4%.)

¹H-NMR (400 MHz, DMSO-d₆) σ: 1.48 (d, 3H, J=6.8 Hz), 2.63 (d, 1H, J=14.0Hz), 2.70 (d, 1H, J=14.0 Hz), 2.90 (d, 1H, J=14.1 Hz), 3.06 (d, 1H,J=14.1 Hz), 4.40 (q, 1H, J=6.8 Hz), 6.91-7.54 (m, 10H).

Example 21 Production of (S)-(−)-1-phenylethylamine salt of(4R)-4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid

(S)-(−)-1-phenylethylamine of 7.12 g (58.7 mmol) was added dropwise tothe solution obtained in Example 20 after the crystal was filtered off,and the mixtured stirred at 25° C. for one hour. The resulting crystalwas filtered off and dried under reduced pressure, to obtain of 23.8 g(53.3 mol) the (S)-(−)-1-phenylethylamine salt of(4R)-4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid. (Yield,81.1%; optical purity, 92.1%.)

Example 22 Production of the ammonium salt of(4S)-4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid

200 ml of water and 18.5 g of aqueous 28% ammonia were added to 21.8 g(51.0 mmol) of the (R)-(+)-1-phenylethylamine salt of(4S)-4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid at 25° C.so as to dissolve the salt, followed by further addition of 200 ml oftoluene and stirring. The aqueous layer obtained by the partition of theresulting layers was heated to 60° C. To the resulting aqueous solutionwas added dropwise 900 ml of 2-propanol over 2 hours. After the aqueous2-propanol solution was cooled to 10° C. over 5 hours, the solution wasstirred at 10° C. for 10 hours. The resulting crystal was filtered anddried under reduced pressure, to obtain 14.75 g of the ammonium salt of(4S)-4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid. (Yield,85.1%; optical purity, 99.0%.)

Melting point; 205° C. (decomposed)

Specific rotation [α]²⁰ _(D)+13.4 (c=1.00, H₂O)

Example 23 Production of the ammonium salt of(4R)-4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid

In the same manner as in the Example described above, 16.2 g theammonium salt of(4R)-4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid wasrecovered of 23.8 g (53.3 mmol) of from the (S)-(−)-1-phenylethylaminesalt of (4R)-4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid.(Yield, 89.3%; optical purity, 99.9%.)

Specific rotation [α]²⁰ _(D)−13.6 (c=1.00, H₂O)

Example 24 Production of (2S,4S) monatin

4.5 g (13.1 mmol) of the ammonium salt of(4S)-4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid wasdissolved in 100 ml of aqueous 28% ammonia, followed by addition of 3.4g of 5% rhodium carbon (50% hydrous product), for reaction at ambienttemperature and a hydrogen pressure of 10 atmospheres (1 MPa). After 24hours, the catalyst was filtered off. The filtrate was concentrated. 40ml of aqueous 90% ethanol was added to the concentrate, for stirring at25° C. for 1.5 hours. The deposited crude crystal was filtered. 40 ml ofaqueous 90% ethanol was added to the crude crystal, and stirred at 25°C. for 1.5 hours. The deposited purified crystal was filtered off anddried under reduced pressure, to obtain 0.57 g (1.84 mmol) of theammonium salt of (2S,4S) monatin. (Yield, 14.1%; optical purity, 99.5%.)

¹HNMR (400 MHz, D₂0) δ: 2.06 (dd, J=11.8, 15.3 Hz, 1H), 2.67 (dd, J=2.0,15.2 Hz, 1H), 3.08 (d, J=14.4 Hz, 1H), 3.28 (d, J=14.4 Hz, 1H) 3.63 (dd,J=2.2, 12.2 Hz, 1H), 7.12-7.16 (m, 1H), 7.20-7.24 (m, 2H), 7.48-7.49 (m,1H), 7.71-7.73 (m, 1H).

Theoretical ESI-MS value C₁₄H₁₆N₂O₅=292.29

Analytical value=291.28 (MH⁻)

Example 25 Production of ammonium salt of (2S,4S) monatin

14.0 g (41.1 mmol) of the ammonium salt of(4S)-4-hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric acid wasdissolved in 120 ml of aqueous 28% ammonia, followed by addition of 7.38g of 5% rhodium carbon (50% hydrous product), for reaction at 25° C. anda hydrogen pressure of 1 MPa. After 24 hours, the catalyst was filteredoff. The filtrate was concentrated. 110 ml of aqueous 88% ethanol wasadded to 17.68 g of the concentrate, for stirring at 25° C. for 19hours. The resulting crude crystal was filtered off and dissolved in 15ml of water, followed by addition of 100 ml of ethanol. After stirringat 25° C. for 1.5 hours, the deposited purified crystals was filteredand dried under reduced pressure, to obtain 4.94 g (16.0 mmol) of theammonium salt of (2S,4S) monatin. (Yield, 39.2%; optical purity, 99.9%.)

Example 26 Production of free form of (2S,4S) monatin

2.22 g (7.18 mmol) of the ammonium salt of (2S,4S) monatin obtained inthe above Example was dissolved in a mixed solvent of 4.5 ml of waterand 4.2 ml (71.8 mmol) of acetic acid, followed by dropwise addition of50 ml of ethanol to the resulting solution at 25° C. over about 3 hours.After another 0.5-hr stirring, the resulting crystal was filtered anddried under reduced pressure, to obtain 1.93 g (6.62 mmol) of (2S,4S)monatin of. (Yield, 92.2%; the ammonium content, 0.19 wt %.)

Comparative Example 1

Using cinchonidine in place of the optically active amine used inExample 20, the same procedures were carried out. The optical purity ofthe resulting crystal was 0%.

Comparative Example 2

L-Lysine was used in place of the optically active amine used in Example20. However, no crystal was obtained.

Comparative Example 3

L-Arginine was used in place of the optically active amine used inExample 20. However, no crystal was obtained.

INDUSTRIAL APPLICABILITY

In accordance with the invention, glutamic acid compounds typicallyincluding monatin useful as sweetener or an intermediate for producingpharmaceutical products can be efficiently produced industrially. Inaccordance with the invention, further, optically active monatin can beefficiently produced industrially.

The present application is based on Japanese application No. 2001-396300(filed Dec. 27, 2001), Japanese application No. 2002-149069 (filed May23, 2002), Japanese application No. 2002-149078 (filed May 23, 2002),and Japanese application No. 2002-182032, filed Jun. 21, 2002, all ofwhich are incorporated herein by reference.

1-32. (canceled)
 33. A compound represented by the formula (4′) or (6′),or a salt thereof:

wherein R² represents a hydrogen atom, an alkyl group, an aryl group oran aralkyl group.
 34. The compound of claim 33, which is represented bythe formula (4′) or a salt thereof.
 35. The compound of claim 33, whichis represented by the formula (6′) or a salt thereof.
 36. The compoundof claim 33, which is a sodium salt, potassium salt, lithium salt,magnesium salt, calcium salt, ammonium salt or dicyclohexylammoniumsalt.
 37. The compound of claim 35, which is a sodium salt, potassiumsalt, lithium salt, magnesium salt, calcium salt, ammonium salt ordicyclohexylammonium salt.
 38. The compound of claim 36, which is asodium salt, potassium salt, lithium salt, magnesium salt, calcium salt,ammonium salt or dicyclohexylammonium salt.
 39. The compound of claim35, wherein R² represents a hydrogen atom.
 40. The compound of claim 35,wherein R² represents an alkyl group.
 41. The compound of claim 35,wherein R² represents an aryl group.
 42. The compound of claim 35,wherein R² represents an aralkyl group.
 43. A process of producing thecompound represented by formula (4′) according to claim 33 or a saltthereof: comprising: cross aldol reacting an indole-3-pyruvic acidrepresented by the formula (1′) or a salt thereof and oxalacetic acidrepresented by the formula (2) or a salt thereof and decarboxylating:

or aldol reacting an indole-3-pyruvic acid represented by the formula(1′) or a salt thereof and a pyruvic acid represented by the formula(2′) or a salt thereof:

to produce the compound represented by formula (4′) or a salt thereof.44. A process of claim 43, comprising aldol reacting theindole-3-pyruvic acid represented by the formula (1′) or a salt thereofand the pyruvic acid represented by the formula (2′) or a salt thereof,wherein the cross aldol reaction is conducted within a range of pH 10 to14.